Fig. 3.

Presentation of 9b protein in SARS-CoV VLPs. Subconfluent 293T cells were transfected with 3 μg of pCAGGS-S, 3 μg of pCAGGS-N(9b⁻), 3 μg of pCAGGS-M, 3 μg of pCAGGS-E and 3 μg of pCAGGS-9b each. At 48 h post transfection, culture medium was harvested and clarified. The released VLPs were first pelleted by centrifugation through a 20% sucrose cushion, and then further purified over a 20–60% sucrose gradient. Twelve fractions were collected from top to bottom; each fraction concentrated by 20% sucrose ultracentrifugation was analyzed using Western blot analysis with anti-S monoclonal antibody (S), anti-N monoclonal antibody (N), anti-HA tag monoclonal antibody (M), and anti-9b monoclonal antibody (9b). β-actin was detected by actin specific polyclonal antibody (Actin). The density of each fraction was measured and is shown.